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RT-RAA detection method for avian infectious bronchitis virus

A technology for RT-RAA and bronchitis, applied in the field of temperature amplification detection, can solve problems such as limitations, inability to reflect the mechanism of genome variation, and inability to detect genotypes

Inactive Publication Date: 2019-09-17
CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, because serotyping only illustrates the differences in IBV neutralizing antigen sites and cannot reflect the variation mechanism of the genome, it is limited in actual detection applications
The genotyping of the S1 gene of IBV using molecular biological methods has been widely used, but as the virus continues to mutate, the existing detection methods based on the S1 gene can no longer detect all genotypes, and it is urgent to establish a method that can detect all genotypes. On-site rapid detection method for clinical detection

Method used

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  • RT-RAA detection method for avian infectious bronchitis virus
  • RT-RAA detection method for avian infectious bronchitis virus
  • RT-RAA detection method for avian infectious bronchitis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Primer and probe design and screening for detection of avian infectious bronchitis virus

[0028] Through the analysis of the S1 gene, the applicant found that because the S1 gene is the typing gene of IBV, it has mutated under the immune pressure of the vaccine and the immune pressure of the avian infectious bronchitis virus vaccine in recent years, so the recent In recent years, avian infectious bronchitis virus has been comprehensively tested. By analyzing the IBV genomes of avian infectious bronchitis virus epidemic strains in recent years, the conserved N gene was finally selected as the target gene, and primers and probes that can detect the currently reported genotypes were designed and screened.

[0029] 306 published N gene sequences containing 9 genotypes of IBV were found by NCBI, and the conserved regions were analyzed according to gene alignment. After homology analysis, the bases located at 751bp-1004bp (reference sequence GenBank accession numb...

Embodiment 2

[0061] Embodiment 2: detection sensitivity and specificity of primer probe

[0062] Detection sensitivity

[0063] Six groups of avian infectious bronchitis virus RNA templates with different concentrations were set up for nucleic acid amplification under the optimal conditions of RT-RAA.

[0064] Extract Avian Infectious Bronchitis Virus RNA according to the instructions of the RNA extraction kit, measure the original concentration of the extracted RNA template, dilute to 1ng / μL according to the ratio, and then dilute to 10 by a 10-fold gradient. -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, 10 -4 ng / μL, 10 -5 ng / μL, 10 -6 ng / μL, take 2 μL respectively as the reaction template, and carry out RT-RAA nucleic acid amplification according to the aforementioned sample loading method.

[0065] The result shows that the primer and probe combination designed by the present invention can guarantee the sensitivity during detection, and the detection sensitivity is RNA final concentration 1...

Embodiment 3

[0068] Embodiment 3: detection application to actual sample

[0069] 1. Sample collection:

[0070] A total of 30 throat swab samples were collected from poultry in a live poultry wholesale market. PBS solution (pH7.0-7.4, 0.01mol / L) was used as the preservation solution (containing penicillin 2000IU / mL, streptomycin 2000IU / mL, nystatin 1000IU / mL, BSA5mg / mL). After collection, the samples were sealed in an incubator with ice and sent to the laboratory for processing or stored at -70°C within 24 hours.

[0071] 2. Sample Preparation

[0072] Place the cotton swab in a centrifuge tube filled with 1 mL of sample preservation solution, vortex and mix well, centrifuge at 10,000 r / min at 4°C for 5 min, and take the supernatant for nucleic acid extraction.

[0073] 3. Nucleic acid extraction

[0074] According to the instructions of the RNA extraction kit, 30 clinical samples to be tested, positive control and negative control RNA were extracted. The extracted RNA must be amplif...

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Abstract

The invention provides an RT-RAA detection method for avian infectious bronchitis virus. The RT-RAA detection method comprises a primer pair for detecting the avian infectious bronchitis virus and a probe set. In the primer pair, the sequence of the forward primer is SEQ ID NO:1, the sequence of the reverse primer is SEQ ID NO:2, and the sequence of the probe is SEQ ID NO:3. The invention provides a method for rapidly detecting the avian infectious bronchitis virus based on molecular biology, so as to realize safe, specific, rapid, sensitive, simple and high-throughput rapid detection of the avian infectious bronchitis virus, thereby making up for the shortcomings of the existing traditional detection technology. In addition, the method is very suitable for on-site rapid detection. Compared with a PCR method, the RT-RAA method performs reaction at a constant temperature, the operation is simple, the shackles of complicated instruments are eliminated, temperature change is not required, and the reaction time is greatly shortened.

Description

technical field [0001] The invention belongs to the technical field of reverse transcription recombinaseaided amplification (Reverse transcription recombinaseaided amplification, RT-RAA) detection technology, and in particular relates to a detection method of avian infectious bronchitis virus RT-RAA. Background technique [0002] Avian infectious bronchitis virus (IBV) can cause an acute, highly contagious viral disease in chickens, namely avian infectious bronchitis. The disease is mainly characterized by coughing, tracheal rales and sneezing. It is one of the most serious viral infectious diseases to the poultry industry in my country. The disease exists in almost all poultry-raising countries in the world. Upward trend. There are many serotypes of IBV, and more than 30 kinds have been reported so far. Different serotype strains have great differences in virulence, pathogenicity and tissue tropism, and there is no or only partial cross-immunity between them. In recent year...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/701C12Q1/6844C12Q2521/507C12Q2521/107C12Q2563/107
Inventor 王素春王楷宬黄保续
Owner CHINA ANIMAL HEALTH & EPIDEMIOLOGY CENT
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